Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, producing light yellow to orange to deep red color. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage. For example, animals do not have the ability to synthesize carotenoids and must obtain these nutritionally important compounds through their dietary sources. Structurally, carotenoids are 40-carbon (C40) terpenoids derived from the isoprene biosynthetic pathway and its five-carbon universal isoprene building block, isopentenyl pyrophosphate (IPP).
Although more than 600 different carotenoids have been identified in nature, only a few are used industrially for food colors, animal feed additives, vitamin A precursors, pharmaceuticals, and cosmetics. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol., 70:181–191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis. At the present time, only a few plants are widely used for commercial carotenoid production.
Carotenoid production by microbial fermentation is a potential way to produce a variety of carotenoids in significant quantities. However, carotenoid production in non-carotenogenic microorganisms requires the ability to genetically engineer genes involved in carotenoid biosynthesis into industrially-useful microorganisms. Recently, carotenoid biosynthesis genes have been isolated from Pantoea stewartii and engineered into recombinant production hosts (WO 02/079395 A2 and commonly owned WO 03/016503 A2 corresponding to U.S. Ser. No. 10/218,118; hereby incorporated by reference). Methods are described for the production of carotenoids such as lycopene, zeaxanthin, canthaxanthin, β-carotene, lutein, and astaxanthin to name a few.
Commercial production of carotenoids not found in nature, or at least ones not naturally produced in commercially-suitable amounts, may also be accomplished by genetic engineering. Production hosts, such as Escherichia coli, can be engineered to produce various novel carotenoids through biochemical pathway engineering. These novel carotenoids may be have superior attributes in comparison to carotenoids currently used in a variety of applications. One such carotenoid is 3,4,3′,4′-tetradehydrolycopene (TDHL).
Most carotenoids exhibit distinct color and can be viewed as natural pigments or colorants. Carotenoids are required elements of aquaculture and are also used in the poultry industry. Salmon and shrimp aquacultures are particularly useful applications as carotenoid pigmentation is critically important for the value of these organisms (Shahidi and Brown, Crit Rev Food Sci, 38(1):1–67 (1998)). Well-known examples of carotenoids used in the aquaculture industry are β-carotene and astaxanthin.
It is also known that carotenoids have utility as intermediates in the synthesis of cosmetics, flavors, and fragrances and compounds with potential electro-optic applications. Tetradehydrolycopene is particularly desirable for electro-optic applications. For example, electrical, optical, and redox characteristics of a polyene are a function of the length of the polyene run. Optical absorption red-shifts as the polyene run becomes longer, and the oxidation and reduction potentials become smaller as the polyene run becomes longer. These properties are believed to approach a limiting value when the polyene has 20 double bonds (W. Vetter et al., in Carotenoids (ed. O. Isler) Birkhäuser, Basel 1971). Tetradehydrolycopene has a run of 15 double bonds extending the entire length of the molecule, while lycopene only has 11 double bonds.
Carotenoids, such as lycopene, are used as antioxidants due to their large number of conjugated double bonds, making inclusion of these compounds in the diet desirable in view of their reported health benefits. Antioxidant potency is attributed to several factors, one being the length of the conjugated polyene chain in acyclic carotenoids (Miller et al., FEBS Letters, 384:240–242 (1996); Albrecht et al., Nature Biotechnology, (18):843–846 (2000)). A carotenoid having a long conjugated double-bond system, such as TDHL, has better antioxidant properties in comparison to carotenoids having a shorter conjugated polyene chain, such as lycopene.
Additionally, the bulk electrical properties of polyenes, such as carotenoids, are determined by the spacing between molecules in the solid state. Lycopene has a sterically bulky pentenyl end group on each side of the molecule, while TDHL does not. This is expected to allow closer interaction of all trans TDHL in the solid state compared to lycopene (Broszeit et al., Liebigs Ann./Recueil, 2205–13 (1997); Heinze et al., J. Solid State Electrochem, 2:102–9 (1998)).
Chemical synthesis of TDHL is not practical and the most direct biological route to this carotenoid species involves the desaturation of phytoene-like substrates by phytoene desaturases. Phytoene desaturase genes have been cloned, expressed, and sequenced from fungal species (Neurospora crassa), cyanobacteria (Synechococcus), bacterial species (Rhodobacter capsulatus, Erwinia uredovora, Erwinia herbicola, and Pantoea stewartii) as well as plant species (Arabidopsis thaliana (Linden et al., supra; Bartley et al., J. Biol. Chem., 265:16020–16024 (1990); Scolnick et al., Plant Physiol., 108: 1343, Bartely et al., Eur. J. Biochem., 265:396–403 (1999); and Hausmann and Sandmann, Fungal Genet. Biol., 30:147–153 (2000)). In addition, oscillaxanthin, a 1,1′-dihydroxy-2,2′-di-β-L-rhamnosyl-1,2,1′,2′-tetrahydro-3,4,3′,4′-tetradehydrolycopene has been characterized from a blue green algae (Arthrospira). Although no genetic data is available, this species presumably contains a gene encoding a phytoene desaturase-type enzyme (Hertzber and Liaaen-Jensen, Phytochemistry, (8):1281–1292 (1969)).
Biological production of TDHL (in trace amounts) in a recombinant host has been reported (Schmidt-Dannert et al., Nature Biotechnology, (18):750–753 (2000); U.S. 2002/0051998 A1)). A mutant phytoene desaturase, synthesized by gene shuffling fragments of the phytoene desaturase genes (crtI) from Erwinia uredovora and Erwinia herbicola, was expressed in E. coli, producing trace amounts of TDHL.
Linden et al. (Z. Naturforsch, (46c):1045–1091 (1991)) expressed the phytoene desaturase (crtI) gene from E. uredovora in E. coli, reporting the production of trace amounts of TDHL. Fraser et al. (J Biol Chem, 267(28):19891–19895 (1992)) reported sporadic production of trace amounts of TDHL when expressing the crtl gene from E. uredovora in E. coli. None of these references teach how to produce TDHL in industrially-suitable amounts in a recombinant host using genes from sources other than E. uredovora or E. herbicola. 
Although small amounts of tetradehydrolycopene have been prepared chemically and trace amounts have been formed in biological systems, no means for economical production of significant amounts of TDHL exists (Hengartner et al., Helvetica Chimica Acta., (75):1848–1865 (1992); Albrecht et al., supra; and Schmidt-Dannert et al., supra). The problem to be solved, therefore, is to provide materials and methods useful for producing industrially-suitable amounts of TDHL in a recombinant production host.
Applicants have solved the stated problem by mutating crtl from Pantoea stewartii and expressing the mutated crtl genes along with other carotenoid biosynthetic enzymes in a recombinant host to produce industrially-suitable amounts of TDHL.